Imaging polycystin-mediated Ca2+ transients in c. elegans

描述（由申请人提供）：多囊肾病（ADPKD）由编码蛋白质多囊蛋白-1和-2的PKD 1和PKD 2基因突变引起。多囊蛋白被认为在肾上皮的初级纤毛中起作用，在那里它们通过流体流动到细胞质钙信号中来阻止纤毛的机械弯曲。假设囊肿发生是由于这种信号通路的中断。然而，多囊蛋白对机械刺激的分子机制还不清楚。线虫C.在线虫中，多囊蛋白直系同源物LOV-1和PKD-2是21个雄性特异性感觉神经元的功能所必需的，所述感觉神经元感测与雌雄同体的接触。多囊蛋白定位于这些细胞树突尖端的初级纤毛。在脊椎动物中，这些蛋白质被认为将纤毛的变形转化为下游的钙信号。由于其复杂和快速的实验可及性，线虫模型具有很大的潜力，探索多囊蛋白信号的分子本质。我们在这方面的应用的目标是开发和实施一个系统，直接测量多囊蛋白依赖的钙信号在体内使用遗传编码的钙指示剂cameleon。在第一个目标中，我们将测量C中的钙瞬变。雄性线虫在真实的时间内对各种刺激做出反应，包括与雌雄同体的接触、人工机械刺激和横向流体流动。通过比较野生型动物和那些携带无效突变的多囊蛋白之间的钙反应，我们将测试的假设，这些反应取决于多囊蛋白的功能。在第二个目标中，我们将应用这种方法来检验假设，两个最近确定的C。在雄性感觉神经元中特异性表达的线虫基因cwp-4和cwp-5在多囊蛋白介导的Ca 2+信号传导中起作用。这些研究将建立C. elegans作为一个独特的系统，在体内遗传和分子分析的分子机制的信号由多囊蛋白在响应机械刺激。

DESCRIPTION (provided by applicant): Polycystic kidney disease (ADPKD) results from mutations in the human PKD1 and PKD2 genes, which encode the proteins polycystin-1 and -2. The polycystins are thought to act in the primary cilia of the renal epithelium, where they transduce mechanical bending of the cilium by fluid flow into cytoplasmic calcium signals. Cystogenesis is hypothesized to result from the disruption of this signaling pathway. However, the molecular mechanisms by which the polycystins respond to mechanical cues are not well understood. In the nematode C. elegans, the polycystin orthologs LOV-1 and PKD-2 are required for the function of twenty-one male-specific sensory neurons that sense contact with hermaphrodites. The polycystins localize to the primary cilia at the dendritic tips of these cells. As in vertebrates, it is thought that these proteins transduce the deformation of cilia into downstream calcium signals. Because of its sophisticated and rapid experimental accessibility, the nematode model has great potential for exploring the molecular nature of polycystin signaling. Our goal in this application is to develop and implement a system to directly measure polycystin-dependent calcium signaling in vivo using the genetically-encoded calcium indicator cameleon. In the first aim, we will measure calcium transients in C. elegans males in real time in response to a variety of stimuli, including contact with hermaphrodites, artificial mechanical stimuli, and transverse fluid flow. By comparing calcium responses between wild-type animals and those carrying null mutations in the polycystins, we will test the hypothesis that these responses depend on polycystin function. In the second aim, we will apply this assay to test the hypothesis that two recently-identified C. elegans genes specifically expressed in male sensory neurons, cwp-4 and cwp-5, have roles in polycystin-mediated Ca2+ signaling. These studies will establish C. elegans as a unique system for the in vivo genetic and molecular analysis of the molecular mechanisms of signaling by the polycystins in response to mechanical stimuli.